ABSTRACT: The interactions between cells and their surrounding extracellular matrix (ECM) regulate various cellular processes, including morphology, differentiation, motility, and fate. The family of adhesion G protein- coupled receptors (aGPCRs), comprising the second largest class of GPCRs with a total of 33 members in the human genome, enables cells to sense their matrix environment. To date, majority of aGPCRs are orphan receptors and each of the few de-orphaned aGPCRs have multiple ligands to mediate cell-cell and ECM interactions. The aGPCR GPR56/ADGRG1 is essential for brain development and postnatal brain wiring (myelination and synaptic formation/refinement). Mutations in GPR56 cause a devastating human brain malformation that is characterized by cortical dysplasia and white matter hypomyelination. Similar to other aGPCRs with known ligands, GPR56 has multiple binding partners, collagen III in the developing cerebral cortex, tissue transglutaminase (TG2) in melanoma, and glycosaminoglycan heparin in cultured cells in vitro. Through unbiased in vitro biotinylation/proteomics approach, our unpublished work demonstrated that TG2 is the ligand of GPR56 in oligodendrocyte precursor cells (OPCs). GPR56 is expressed in multiple cell types in the developing brain, including radial glial cells, neural progenitor cells, astrocytes, OPCs, and microglia. We hypothesize that GPR56 carries out distinct developmental tasks in different cell types via differential signaling in response to varied ligands to mediate these effects. To begin to test this hypothesis, we propose to generate genetic tool kits that will allow us to eventually carry out in vivo biotinylation proteomics to reveal GPR56 ?interactome? landscape.